Biological switches control cell-fate decisions across biological kingdoms. Many of these cell-fate decisions are controlled by complex transcriptional networks, consisting of unique combinations of transcriptional regulators and their respective cis-regulatory targets. An example of such a network is that which controls the white-opaque phenotypic switch in the human fungal pathogen, Candida albicans. The white-opaque switch gives rise to two distinct cell types – white and opaque – which differ in their mating competence, adaptation to distinct environmental niches, host immune cell evasion, and morphological characteristics at the single-cell and colony level. Each cell type can be heritably maintained through many generations and stochastic or environmentally induced switching can lead to a transition between these two phenotypic states. Switching between these two heritably maintained transcriptional programs occurs without any changes to the primary sequence of the genome, and thus fits the classic definition of an epigenetic switch. The ability of C. albicans to readily switch between the white and opaque phenotypes is controlled by a “core” circuit of eight transcription factors (TFs) that is centered around the opaque-specific master regulator Wor1 (White-Opaque Regulator 1), however, recent work has identified a set of nineteen new regulators that impinge upon the switch. In this work, we characterize these newly identified TFs and their interactions with the previously defined “core” regulators as well as determine how these new regulators affect the establishment and maintenance of the white-opaque switch.

The human body is made of many different cell types, the vast majority of which contain the same underlying genetic information. This singular genetic blueprint gives rise to unique transcriptional programs that specify patterns of gene expression and establish numerous distinct cell types, and chromatin plays an important role in this process. Research in the field of chromatin biology has historically separated the two main constituents of chromatin: nucleosomes, and the relatively smaller transcription factors (TFs). Recently, however, a growing body of evidence shows that it is the interplay between TFs and nucleosomes, two opposite but interconnected forces, that drives chromatin mediated mechanisms in a yin and yang fashion. Despite the progress made thus far, we still lack a clear understanding of the mechanisms through which chromatin regulates cell type establishment and heritability. In this dissertation, I examined how the interplay between TFs and nucleosomes regulates phenotypic switching between two distinct cell types referred to as white and opaque in the diploid polymorphic fungus Candida albicans. The white-opaque switch is observed in C. albicans and several closely related species but is absent in other common model yeasts like Saccharomyces cerevisiae. The two cell types are established and maintained by distinct transcriptional programs, leading to differences in metabolic preferences, mating abilities, cellular morphologies, responses to environmental signals, interactions with the host immune system and differential expression of ~20% of the transcriptome. Remarkably, the level of connectivity of the opaque cell transcriptional circuit closely resembles the circuits that control stem cell maintenance and differentiation in humans, indicating that the white-opaque switch represents an attractive and relatively “simple” model system to investigate how the interplay between TFs and chromatin structure regulates heritable cell fate decisions in higher eukaryotes. For this work, I adapted several next-generation sequencing (NGS) based assays to examine chromatin structure in C. albicans, one of which I describe in Chapter 3. Next, I used these genome-wide approaches to show that the opaque cell “master regulator” Wor1 establishes opaque-specific chromatin structures, some of which are required for heritable maintenance of the opaque transcriptional program (Chapter 2). All-in-all, I show that, in addition to Wor1, there are more TFs (Ndt80 and Wor2) with specialized ability to target many Nucleosome-Free Regions (NFRs)—compact chromatin structures that are observed to be inaccessible by ATAC-seq assays—and effect the expression of a large number of genes. Since this specialized ability to organize chromatin is observed throughout the genome, these types of chromatin organizing TFs are often recognized to serve as master regulators that are critical for developmental processes and heritable cell fate decisions. The work presented in this thesis will serve as a foundation to further investigate how the interplay between chromatin organizing TFs and dynamic nucleosomes regulates developmental processes and heritable cell fate decisions in C. albicans, and may lead to insights into how these processes regulate cellular differentiation in higher eukaryotes.